Tobacco smoking is responsible for more than 400,000 deaths annually in the United States. Nicotine is the major addictive agent in tobacco, the key ingredient that maintains smoking behavior. Individual differences in nicotine metabolism affect smoking behavior and potentially nicotine addiction. Nicotine is primarily metabolized by 5'-oxidation. P450 2A6 is the major hepatic catalyst of this reaction, and we recently reported that P450 2A13, a lung P450, is also a good catalyst. An unexpected and exciting outcome of these studies was that nicotine was a mechanism-based (suicide) inhibitor of P450 2A6 and P450 2A13. Determining the mechanism of this inhibition is the primary goal of this grant. However, to begin to investigate the impact of nicotine-mediated inactivation on whole tissue metabolism, we will study P450 2A3 inactivation in the perfused rat lung. P450 2A3 (89% identical to P450 2A13) is an excellent catalyst of nicotine metabolism. Our hypothesis is that inactivation of P450 2A6, P450 2A13 and P450 2A3 occurs through a common mechanism and that any differences in inactivation are due to differences in the specificity and catalytic efficiency of these enzymes. Nicotine 5'-oxidation, as well as minor pathways of 2'- and methyl oxidation, generates reactive iminium ions, all possible inactivating molecules. However, preliminary data support a metabolite of the ?5'(1') iminium ion, as the inactivating species. This grant will investigate what pathway of nicotine metabolism is responsible for enzyme inactivation and how P450 2A3 inactivation affects the metabolic capacity of the rat lung. The Specific aims are: 1. To characterize the secondary products of nicotine metabolism and determine their role in inactivation. 2. To determine if modification of the heme and/or apoprotein occurs during nicotine-mediated inactivation of P450 2A enzymes, both Mass Spectrometry and NMR analyses will be carried out. 3. To establish the rat lung as a model system in which to investigate potential in vivo effects of nicotine-mediated P450 2A inactivation. The characterization of nicotine-mediated inactivation of P450 2A6 and 2A13 is critical to an accurate and complete understanding of nicotine metabolism in smokers. In addition, identifying the mechanism by which this occurs will aid in the design of nicotine analogs as enzyme inhibitors and potential medications for treatment of nicotine addiction. Also of key importance, is that the inactivation of P450 2A enzymes will impact the metabolism of the tobacco specific carcinogens, NNK and NNN.